The relationship between structure and function of macromolecules of fundamental importance in the understanding of biological systems. These relationships are important to understanding, for example, the functions of enzymes, structure of signalling proteins, ways in which cells communicate with each other, as well as mechanisms of cellular control and metabolic feedback.
Genetic information is critical in continuation of life processes. Life is substantially informationally based and its genetic content controls the growth and reproduction of the organism. The amino acid sequences of polypeptides, which are critical features of all living systems, are encoded by the genetic material of the cell. Further, the properties of these polypeptides, e.g., as enzymes, functional proteins, and structural proteins, are determined by the sequence of amino acids which make them up. As structure and function are integrally related, many biological functions may be explained by elucidating the underlying structural features which provide those functions, and these structures are determined by the underlying genetic information in the form of polynucleotide sequences. In addition to encoding polypeptides, polynucleotide sequences can also be specifically involved in, for example, the control and regulation of gene expression.
The study of this genetic information has proved to be of great value in providing a better understanding of life processes, as well as diagnosing and treating a large number of disorders. In particular, disorders which are caused by mutations, deletions or repeats in specific portions of the genome, may be readily diagnosed and/or treated using genetic techniques. Similarly, disorders caused by external agents may be diagnosed by detecting the presence of genetic material which is unique to the external agent, e.g., bacterial or viral DNA.
While current genetic methods are generally capable of identifying these genetic sequences, such methods generally rely on a multiplicity of distinct processes to elucidate the nucleic acid sequenes, with each process introducing a potential for error into the overall process. These processes also draw from a large number of distinct disciplines, including chemistry, molecular biology, medicine and others. It would therefore be desirable to integrate the various process used in genetic diagnosis, in a single process, at a minimum cost, and with a maximum ease of operation.
Interest has been growing in the fabrication of microfluidic devices. Typically, advances in the semiconductor manufacturing arts have been translated to the fabrication of micromechanical structures, e.g., micropumps, microvalves and the like, and microfluidic devices including miniature chambers and flow passages.
A number of researchers have attempted employ these microfabrication techniques in the miniaturization of some of the processes involved in genetic analysis in particular. For example, published PCT Application No. WO 94/05414, to Northrup and White, incorporated herein by reference in its entirety for all purposes, reports an integrated micro-PCR apparatus for collection and amplification of nucleic acids from a specimen. However, there remains a need for an apparatus which combines the various processing and analytical operations involved in nucleic acid analysis. The present invention meets these and other needs.
The present invention generally provides miniature integrated fluidic systems for carrying out a variety of preparative and analytical operations, as well as methods of operating these systems and methods of using these systems. In a first aspect, the present invention provides a miniature fluidic system which comprises a body having at least first and second chambers disposed therein. Each of these first and second chambers has a fluid inlet and is in fluid connection. At least one of these first and second chambers is a hybridization chamber for analyzing a component of a fluid sample. The hybridization chamber includes a polymer array which has a plurality of different polymer sequences coupled to a surface of a single substrate, each of the plurality of different polymer sequences being coupled to the surface in a different, known location. The system further includes a sample inlet, fluid connected to at least one of the first and second chambers, for introducing a fluid sample into the system, and a fluid transport system for moving a fluid sample from the first chamber to the second chamber.
In a preferred aspect, the fluid direction system comprises a pneumatic manifold for applying a differential pressure between the first chamber and the second chamber, to move said fluid sample from the first chamber to the second chamber.
In a related aspect, the present invention provides a miniature fluidic system, which is substantially the same as that described above, except that in place or in addition to a hybridization chamber, the system comprises a separation channel for separating a component of said fluid sample. The separation channel is fluidly connected to at least one of the chambers and includes at least first and second electrodes in electrical contact with opposite ends of the separation channel for applying a voltage across said separation channel.
Similarly, in an additional aspect, the present invention provides a substantially similar fluidic system as described, except where at least one of the chambers comprises an in vitro transcription reaction chamber, the in vitro transcription reaction chamber having an effective amount of an RNA polymerase and four different nucleoside triphosphates, disposed therein.
Further, the system may comprise a body wherein at least one of the chambers is a cell lysis chamber which includes a cell lysis system, for lysing cells in said fluid sample.
In a still further related aspect, at least one of the chambers may be a nucleic acid purification chamber, for separating nucleic acids in said fluid sample from other contaminants in said fluid sample.
The present invention also privates a miniature fluidic system whicn comporises a differential pressure delivery system for transporting fluids through the system. In particular, in one aspect, the present invention provides a miniature fluidic system, which includes a body having at least a first reaction chamber fluidly connected to a second reaction chamber by a fluid passage. The system also includes a sample inlet, fluidly connected to the first chamber, for introducing a fluid sample into the system. The system further includes a differential pressure delivery system for maintaining the first chamber at a first pressure and the second chamber at a second pressure, wherein the first pressure is greater than ambient pressure and the second pressure is greater than said first pressure. When the second chamber is brought to ambient pressure, the first pressure forces a liquid sample in the first chamber into the second chamber.
In an alternate aspect, the fluidic system employs a differential pressure delivery source for maintaining the first chamber at a first pressure and the second chamber at a second pressure, where the second pressure is less than ambient pressure and the first pressure is less than the second pressure. When the first chamber is brought to ambient pressure, the second pressure draws a liquid sample in the first chamber into the second chamber.
The present invention also provides methods of directing, controlling and manipulating fluids in miniature or micro-fluidic systems.
For example, in one aspect, the present invention provides a method for directing a fluid sample in a miniature fluidic system which comprises providing a microfabricated device having at least first and second chambers disposed therein, wherein each of said at least first and second chambers is in fluid connection with a common chamber or channel, has at least first and second controllable valves disposed across said fluid connection, respectively, and includes at least one vent. The method comprise applying a positive pressure to the common chamber or channel. The at least first controllable valve is selectively opened, whereby the positive pressure forces the fluid sample from the common chamber or channel into the first chamber.
The method may further comprise applying a positive pressure to the first chamber and selectively opening the least first controllable valve, whereby the positive pressure forces said fluid sample from the least first chamber into the common chamber or channel.
The present invention also provides methods of mixing at least two discrete fluid components in a microfabricated fluidic system. Specifically, the method comprises providing a microfabricated channel having a vent disposed at an intermediate location in said channel. Typically, the vent includes a gas permeable, fluid barrier disposed across the vent. At least two discrete fluid components are then introduced into the channel separated by a gas bubble. Upon flowing the at least two fluid components past the vent, the bubble will exit the vent, allowing the at least two fluid components to mix.
The present invention also provides methods of repeatedly measuring a known volume of a fluid in a miniature fluidic system. In particular, the method comprises providing a microfabricated device having at least first and second chambers disposed therein, wherein the at least first and second chambers are in fluid connection, and wherein at least one of the chambers is a volumetric chamber having a known volume. The volumetric chamber is filled with the fluid to create a first aliquot of the fluid. This aliquot is then transported to the at least second chamber and the filling and transporting steps are repeated.